To complement studies being conducted by NCI investigators to understand the cyclic AMP signaling cascades that occur during chemotaxis in Dictyostelium discoideum, we are determining the high-resolution three-dimensional arrangement of vesicles that are observed by fluorescence light microscopy of GFP-labeled migrating D. discoideum. Cells were introduced into plastic capillary tubes or deposited onto sapphire discs and were allowed to attach. The specimens were then frozen at 2000 atmospheres in a high-pressure freezing machine to preserve their cellular ultrastructure. The frozen blocks were freeze-substituted in acetone, embedded in plastic, sectioned and stained and imaged by transmission electron microscopy. Serial sectioning is being performed to visualize ultrastructural differences between the anterior and posterior regions of the migrating cells and to compare with structure observed by light microscopy. In addition, cells are being immunolabeled with the aim of localizing adenylyl cyclase at the ultrastructural level.